1. Field of the Invention
This invention relates to recombinant DNA techniques for producing human lysozyme. More particularly, it relates to the expression of the human lysozyme gene, recombinant plasmids, transformed cells, and their products.
2. Description of the Prior Art
Lysozyme is a comparatively small enzyme protein with a molecular weight of approx. 14,000. Lysozyme is distributed in living tissue and is believed to play a role as a defensive substance against bacterial infections by dissolving various bacteria. The method of lysozyme function is thought to involve hydrolysis of polysaccharide of the bacterial cell wall by .beta.-glucosidase activity. A good deal of lysozyme is contained in hen egg white and lysozyme with high purity can be isolated therefrom comparatively easily. Lysozyme is thus added to cheese, sausage and marine products for their preservation or used for the purpose of converting bovine milk to human maternal milk [Katsuya Hayashi and Taiji Imoto, "Lysozyme", Nankodo, Japan (1974)]. Further, as a medicinal agent, lysozyme is used as a hemostatic, anti-inflammation, tissue regeneration, anti-tumor etc. agent [see for instance "Saikin-no-Shinyaku" (New Drugs in Japan) vol. 34, 107, Yakuji Nippo, Tokyo, Japan, 1983) and it is also on sale as anti-infla-mmatory enzyme agent.
When the lysozyme derived from hen egg white is used for medical purposes, sensitive symptoms such as rash, redness, etc. often result. These are generally regarded as a side effect of the immune response to the presence of a foreign protein. To overcome the above-mentioned disadvantages, the present inventors began studying to establish a means for the mass production of human lysozyme.
Amino acid sequence of human lysozyme is known, and consists of 130 amino acids ["Biochemical Data Book" 1, 189 (1979), editted by Japan Biochemical Association]. On the basis of the amino acid sequence, a DNA fragment coding for the human lysozyme is chemically synthesized [Ikehara, M. et al, Chem. Pharm. Bull. 34, 2202 (1986)].
For the production of human lysozyme using a recombinant DNA technique, Muraki et al tried to express it in E. coli but failed to obtain active human lysozyme with such an expression system [Muraki, M. et al, Agric. Biol. Chem. 50, 713 (1986)].
An active human lysozyme has been obtained by secretion using a yeast [Jigami et al, Summary of Japanese Agricultural Chemistry Association 1986, p.343 (1986)]. But the yield is very low, i.e. only 600 .mu.g/liter. According to Jigami et al, a cDNA fragment coding for a signal peptide having information for extracellular secretion of a desired protein is used in combination with a DNA fragment coding for a human lysozyme for the expression of the human lysozyme. Jigami et al uses a cDNA fragment (natural type) coding for a signal peptide of egg white lysozyme [The signal peptide has the same sequence as that disclosed in Pro. Natl. Acad. Sci. U.S.A. 77, 5759-5763 (1980)]. With such a combination, however, the lysozyme is secreted extracellularly from yeast cells in a very small amount as described above.